The goal of this proposal is to discover novel genetic variants that regulate sensitivity to an addictive drug, cocaine. Discovery of novel genes and alleles that mediate addiction will lend itself to discovery of mechanistic components in addiction pathways that can be targeted for treatment and intervention. We have taken a forward genetic mutagenesis approach to discover variants that regulate acute response to cocaine, hyperactivity and anxiety, all predictors of addictive behavior. Mutagenesis based screening in model animals is completely unbiased and has been bedrock in biology for over 50 years. We have conducted one of the largest screens to date for cocaine response and open field behaviors. From this screen of over 17,000 mice we have a collection of ten mutants and have genetically mapped three of these mutants to large chromosomal intervals. Now, we propose to leverage next-generation sequencing technology to find the exact mutation responsible for the phenovariance in these mutants. We will also carry our RNA-Seq to understand the transcriptomic program that is perturbed in these mutants. We will carry out an integrated multistep next- generation sequencing approach utilizing exome, whole genome, and RNA sequencing to study these mutants. Successful completion of this project will lead to detection of viable causal variants for addiction liability and identification of the pathway perturbed by th variant. This project has significant potential for discovery of novel genes, alleles, and pathways that regulate cocaine responses. From this information we will be able to identify new neural substrates of addiction, discover novel molecular components and establish a better mechanistic understanding of cocaine response and its proximity to potential druggable targets. We will also provide the addiction research community with new mouse models for altered drug response.